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Inhibition peptide of hepatatitis B virus polyzymoprotein YMDD functional region and its application

A technology of hepatitis B virus and polymerase protein, which is applied in the fields of molecular biology and biochemistry of biotechnology, and can solve the problems of virus resistance, inability to completely cure hepatitis B, inhibit HBVcccDNA synthesis, and incapable of curing hepatitis B.

Inactive Publication Date: 2006-06-14
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The fundamental reason why hepatitis B cannot be completely cured is that the currently used antiviral drugs cannot inhibit the synthesis of HBV cccDNA in the nucleus of liver cells
HBV reverse transcriptase inhibitors block most new HBV DNA synthesis but have little effect on the cccDNA pool in the nucleus of up to 50 or more copies
This is also the reason why nucleoside analogues such as lamivudine (Lamivudine, 3TC) currently used clinically cannot cure hepatitis B.
Moreover, it has been found that some patients have mutations in the functional region of the viral polymerase P protein YMDD after treatment with lamivudine, resulting in virus resistance to lamivudine

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Screening of inhibitory peptides for the YMDD functional region of hepatitis B virus polymerase protein

[0023] (1) Synthesis of short peptides in the YMDD functional region of the hepatitis B virus polymerase protein: Designed according to the HBV ayw genotype sequence (genbank access no: U95551), synthesized short peptides in Hangzhou Zhongpi Biochemical Co., Ltd. The sequence is: Arg Arg Ala PhePro His Cys Leu Ala Phe Ser Try Met Asp Asp Val Val Leu Gly Ala. The purity of the short peptide is more than 85%, and the solubility is more than 0.5 mg / ml.

[0024] (2) Screening of phage peptide library

[0025] 1. The first day:

[0026] 1.1. With 0.1M NaHCO 3 (PH 8.6) Prepared target molecule solution: Hepatitis B virus polymerase protein YMDD functional region short peptide is made into a solution of 150μg / ml, first dissolved with DMSO, and then 0.1M NaHCO 3 (PH 8.6) dissolved.

[0027] 1.2. Coating: Add 150μl of target molecule solution to each well, and vortex r...

Embodiment 2

[0085] Example 2 Experiment of inhibitory peptide on the inhibitory effect of hepatitis B virus

[0086] (1) Cell culture

[0087] 1. Cultivation of 2.2.15 cells 2.2.15 cells are hepatocellular carcinoma cell lines transfected with full HBV genes, containing polyploidy of complete HBV genes, capable of transcribing and translating HBV genes and producing and secreting HbsAg, HbeAg, HBV DNA and HBV Particles. It was cultured under pressure with RPMI1640 medium, 10% newborn calf serum, 400mg / L G418. 2.2.15 cells press 2×10 6 / Plate inoculated on a 60mm cell culture plate. The experimental group consists of synthetic peptides (Trp Try Thr Asn Asn Ser Thr (HBV YMDD functional area inhibitory peptide 1#), Gly Pro Phe Asn Asn Pro Pro (HBV YMDD functional area inhibitory peptide 2#), Gly Trp Leu Pro ProPro Asp( HBV YMDD functional area inhibitory peptide 3#), synthesized by Hangzhou Zhongtide Biochemical Co., Ltd., add 30μl inhibitory peptide solution to each plate. In the lamivudine con...

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PUM

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Abstract

The inhibiting peptide of hepatitis B virus polymerase protein YMDD function zone and application belong to the field of molecular biology and biological chemistry of biological technology. Said invention uses short peptide of hepatitis B virus polymerase protein YMDD function zone as target position, and utilizes bacteriophage surface spreading technique to screen the peptide which can be combined with specificity of the described target position. The amino acid sequences of three inhibiting peptides screened by said invention respectively are Trp Try Thr Asn Asn Ser Thr; Gly Pro Phe Asn Asn Pro Pro; and Gly Trp Leu Pro Pro Pro Asp. The tests show that said three inhibiting peptides have obvious inhibiting action fro DNA of HBV.

Description

Technical field [0001] The present invention belongs to the fields of molecular biology and biochemistry of biotechnology, and relates to inhibitory peptides of the YMDD functional region of hepatitis B virus polymerase protein screened from a phage peptide library, which can inhibit hepatitis B virus replication and is suitable for anti-B Research on hepatitis virus drugs and molecular virology of hepatitis B virus. Background technique [0002] Chronic hepatitis B is caused by hepatitis B virus (HBV), a common disease that seriously endangers human health. The prevention and treatment of chronic hepatitis B is a global public health problem, which has attracted the attention of countries all over the world. . The development of effective anti-hepatitis B virus therapeutic drugs is a major issue that needs to be solved urgently. [0003] In the HBV replication cycle, the HBV covalently closed circular DNA (cccDNA) molecule in the hepatocyte nucleus is the synthesis template of H...

Claims

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Application Information

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IPC IPC(8): C07K7/06A61K38/08A61P31/12
Inventor 朱海红周林福贾红宇陈智
Owner ZHEJIANG UNIV
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